As a model for functional-biological genome analyses, Giardia provides unique perspectives on the origins of eukaryotic cells. This proposal belongs to an Interactive Research Project Grant (IRPG), which will synergize strongly with an R01 "Giardia: A Model for Ancient Eukaryote Genome Analysis" (Mitchell Sogin PI, projected start date Feb. 1. 1998), whose overall goal is to obtain accurate sequence and mapping information for at least 95 percent of this small, but evolutionarily crucial genome. Our tests of inferred function of a carefully selected sample will greatly extend and validate the sequence information. The major goal unifying these biological analyses is to obtain insights into the genetic innovations that led to compartmentalization of important functions in eukaryotic cells with the following Specific Aims: The view that Giardia is "pre-mitochondrial" has been challenged by our finding of three mitochondrial-like genes whose expression, location and activities we will analyze in Specific Aim 1. In Specific Aim 2, we will gain insights into the evolution of eukaryotic endomembrane systems by analyses of three giardial protein disulfide isomerases that have both pro- and eukaryotic properties. Since the organization of genetic material is increasingly complex in eukaryotes, in Specific Aim 3, we will analyze a model region of very high transcript density to elucidate strategies that Giardia can use to efficiently exploit its limited genome size. In Specific Aim 4, we request the capability to analyze novel genes chosen for their power to extend our understanding of the complexity of eukaryotic cellular compartments or of giardial pathogenesis. This project will provide novel insights into the evolution of basic eukaryotic cellular mechanisms, as well as specific information on an important environmentally transmitted infectious agent. It has the unique potential to reveal biological innovations reflective of the prokaryotic/eukaryotic divergence.